Process for the biological production of 1,3-propanediol with high titer Number:7,067,300 from the United States Patent and Trademark Office (PTO) owispatent

Title: Process for the biological production of 1,3-propanediol with high titer

Abstract: The present invention provides an improved method for the biological production of 1,3-propanediol from a fermentable carbon source in a single microorganism. In one aspect of the present invention, an improved process for the conversion of glucose to 1,3-propanediol is achieved by the use of an E. coli transformed with the Klebsiella pneumoniae dha regulon genes dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, and orfZ, all these genes arranged in the same genetic organization as found in wild type Klebsiella pneumoniae. In another aspect of the present invention, an improved process for the production of 1,3-propanediol from glucose using a recombinant E. coli containing genes encoding a G3PDH, a G3P phosphatase, a dehydratase, and a dehydratase reactivation factor compared to an identical process using a recombinant E. coli containing genes encoding a G3PDH, a G3P phosphatase, a dehydratase, a dehydratase reactivation factor and a 1,3-propanediol oxidoreductase (dhaT). The dramatically improved process relies on the presence in E. coli of a gene encoding a non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol.

Patent Number: 7,067,300 Issued on 06/27/2006 to Emptage,   et al.

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Inventors:	Emptage; Mark (Wilmington, DE); Haynie; Sharon L. (Philadelphia, PA); Laffend; Lisa A. (Claymont, DE); Pucci; Jeff P. (Pacifica, CA); Whited; Gregory Marshall (Belmont, CA)
Assignee:	E. I. du Pont de Nemours and Company (Wilmington, DE)
Appl. No.:	277249
Filed:	October 21, 2002

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Related U.S. Patent Documents

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	Application Number	Filing Date	Patent Number	Issue Date
	09641652	Aug., 2000	6514733
	60149534	Aug., 1999

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Current U.S. Class:	435/252.3 ; 435/252.7; 435/252.8; 435/252.9; 435/253.3; 435/253.5; 435/254.11; 435/254.21; 435/254.23; 435/254.3; 435/254.8; 435/255.4; 536/23.1; 536/23.2
Current International Class:	C12N 1/20 (20060101); C07H 21/02 (20060101); C12N 1/14 (20060101)
Field of Search:	435/189,252.33,252.7,252.9,254.3,254.21,254.23,252.31,252.8,252.34,253.5,254.8,155 536/23.2,23.1

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References Cited [Referenced By]

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U.S. Patent Documents

	5686276		November 1997		Laffend et al.
	6013494		January 2000		Nakamura et al.

Foreign Patent Documents

	3734 764		May., 1989		DE
	373 230		Feb., 1993		EP
	WO 9821339		May., 1998		WO
	WO9821341		May., 1998		WO
	WO9928480		Jun., 1999		WO

Other References

Daniel et al., FEMS Microbiol. Rev. 22, pp 553-566 Biochemisrty of coenzyme B12-dependent glycerol and diol dehydratases and organization of the encoding genes. (1999). cited by other . Toraya and Mori, J. Biol. Chem. vol. 274(6),pp 3372-3377 A reactivating factor for Coenzyme B12-dependent Diol Dehydratase (1999). cited by other . GenBank AF026270. cited by other . Wang et al., J. Bact., vol. 176(22), pp 7091-7095 Cloning, Sequence, and Disruption of the Saccharomyces diastaticus DAR1 Gene Encoding a Glycerol-3-Phosphate Dehydrogenase (1994). cited by other . Larason et al., Mol. Microbiol. vol. 10(5), pp, 1101-1111 A gene encoding sn-glycerol 3-phosphate dehydrogenase(NAD+) complements an osmosensitive mutant of Saccharomyces cerevisiae (1993). cited by other . Albertyn et al., (Mol. Cell. Biol., vol. 14(6),pp 4135-4144 GPD1, Which Encodes Glycerol-3-Phosphate Dehydrogenase, Is Essential for Growth under Osmotic Stress in Saccharomyces cerevisiae, and Its Expression Is Regulated by the High-Osmolarity Glycerol Response Pathway, (1994). cited by other . Norbeck et al., J. Bio. Chem., vol. 271(23) , pp 13875-13881 Purification and Characterization of Two Isoenzymes of DL-Glycerol-3-phosphatase from Saccharomyces cerevisiae. (1996). cited by other . Veiga DA Cunha et al., J. Bacteriology,vol. 174(3),pp 1013-1019 Sugar-Glycerol Cofermentations in Lactobacilli: the Fate of Lactate (1992). cited by other . Stieb et al., Arch. Microbiol. 140, pp 139-146 A new 3-hydroxybutyrate fermenting anaerobe, Ilyobacter polytropus.gen.nov.sp.nov., possessing various fermentation pathways (1984). cited by other . Tong et al., Appl. Biochem. Biotech. vol. 34/35, pp 149-159 Enhancement of 1,3-Propanediol Production by Cofermentation on Escherichia coli Expressing Klebsiella pneumoniac dha Regulon Genes (1992). cited by other . Tong, Ph.D., Thesis, University of Wisconsin-Madison (1992). cited by othe- r . Saint-Amans et al., Biotechnology Ltrs. vol. 16(8), pp 831-836 High Production of 1,3-Propanediol from Glycerol by Clostridium Butyricum VPI 3266 in a simply controlled Fedbatch system (1994). cited by other . Abbad-Andaloussi et al., Appl. Environ. Microbiol., vol. 61(12), pp 4413-4417 Isolation and Characterization of Clostridium butyricum DSM 5431 Mutants with Increased Resistance to 1,3-Propanediol and Alered Production of Acids. (1995). cited by other . Homann et al., Appl. Bicrobiol., Biotechnol. vol. 33, pp 121-126 Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains. (1990). cited by other . Blattner et al. Escherichia coli K-12 MG1655 section 273 of 400 of the complete genome XP002162541. cited by other . Bouvet et al., Taxonomic diversity of anaerobic glycerol dissimilation in the Enterobacteriaceae, Research in Microbiology vol. 146, No. 4, 1995 pp 279-290 XP000982719. cited by other . Skraly et al., Construction and characterization of a 1,3-propanediol operon, Applied and Environmental Microbiology. US, Washington, D.C. vol. 64, No. 1, Jan. 1998 pp 98-105, XP002134649. cited by other.

Primary Examiner: Prouty; Rebecca E.
Assistant Examiner: Walicka; Malgorzata

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Parent Case Text

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CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. patent application Ser. No. 09/641,652 filed Aug. 18, 2000, now U.S. Pat. No. 6,514,733, which claims benefit of provisional application 60/149,534 filed Aug. 18, 1999.

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Claims

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What is claimed is:

1. A microorganism transformed with a chimeric gene comprising an isolated nucleic acid fragment encoding a polypeptide with non-specific catalytic activity for the conversion of 3-hydroxypropionaldehyde to 1,3-propanediol operably linked to suitable regulatory sequences, wherein the isolated nucleic acid fragment is selected from the group consisting of: (a) an isolated nucleic acid fragment encoding the amino acid sequence of SEQ ID NO:57; (b) an isolated nucleic acid fragment encoding a polypeptide of at least 387 amino acids having at least 95% identity with the amino acid sequence of SEQ ID NO:57; (c) an isolated nucleic acid fragment that hybridizes with (a) or (b) under hybridization conditions of 0.1.times.SSC, 0.1% SDS, 65.degree. C. and washed with 2.times.SSC, 0.1% SDS followed by 0.1.times.SSC, 0.1% SDS; and (d) an isolated nucleic acid fragment that is complementary to (a), (b) or (c); wherein the transformed microorganism is further transformed with genes encoding a glycerol or diol dehydratase enzyme, and is selected from the group consisting of Citrobacter, Enterobacter, Clostridium, Kiebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Salmonella, Bacillus, Streptomyces, Escherichia, and Pseudomonas.

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Description

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FIELD OF INVENTION

This invention comprises process for the bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism.

BACKGROUND

1,3-Propanediol is a monomer having potential utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds.

A variety of chemical routes to 1,3-propanediol are known. For example ethylene oxide may be converted to 1,3-propanediol over a catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid, by the catalytic solution phase hydration of acrolein followed by reduction, or from compounds such as glycerol, reacted in the presence of carbon monoxide and hydrogen over catalysts having atoms from group VIII of the periodic table. Although it is possible to generate 1,3-propanediol by these methods, they are expensive and generate waste streams containing environmental pollutants.

It has been known for over a century that 1,3-propanediol can be produced from the fermentation of glycerol. Bacterial strains able to produce 1,3-propanediol have been found, for example, in the groups Citrobacter, Clostridilm, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. In each case studied, glycerol is converted to 1,3-propanediol in a two step, enzyme catalyzed reaction sequence. In the first step, a dehydratase catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA) and water, Equation 1. In the second step, 3-HPA is reduced to 1,3-propanediol by a NAD.sup.+-linked oxidoreductase, Equation 2. The 1,3-propanediol is not metabolized further and, as a result, Glycerol.fwdarw.3-HPA+H.sub.2O (Equation 1) 3-HPA+NADH+H.sup.+.fwdarw.1,3-Propanediol+NAD.sup.+ (Equation 2) accumulates in the media. The overall reaction consumes a reducing equivalent in the form of a cofactor, reduced .beta.-nicotinamide adenine dinucleotide (NADH), which is oxidized to nicotinamide adenine dinucleotide (NAD.sup.+).

In Klebsiella pneumonia, Citrobacter freundii, and Clostridium pasteurianum, the genes encoding the three structural subunits of glycerol dehydratase (dhaB1-3 or dhaB, C and E) are located adjacent to a gene encoding a specific 1,3-propanediol oxidoreductase (dhaT) (see FIG. 1). Although the genetic organization differs somewhat among these microorganisms, these genes are clustered in a group which also comprises orfX and orfZ (genes encoding a dehydratase reactivation factor for glycerol dehydratase), as well as orfY and orfW (genes of unknown function). The specific 1,3-propanediol oxidoreductases (dhaT's) of these microorganisms are known to belong to the family of type III alcohol dehydrogenases; each exhibits a conserved iron-binding motif and has a preference for the NAD.sup.+/NADH linked interconversion of 1,3-propandiol and 3-HPA. However, the NAD.sup.+/NADH linked interconversion of 1,3-propandiol and 3-HPA is also catalyzed by alcohol dehydrogenases which are not specifically linked to dehydratase enzymes (for example, horse liver and baker's yeast alcohol dehydrogenases (E.C. 1.1.1.1)), albeit with less efficient kinetic parameters. Glycerol dehydratase (E.C. 4.2.1.30) and diol [1,2-propanediol] dehydratase (E.C. 4.2.1.28) are related but distinct enzymes that are encoded by distinct genes. Diol dehydratase genes from Klebsiella oxytoca and Salmonella typhimurium are similar to glycerol dehydratase genes and are clustered in a group which comprises genes analogous to orfX and orfZ (Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999); Toraya and Mori, J. Biol. Chem. 274, 3372 (1999); GenBank AF026270).

The production of 1,3-propanediol from glycerol is generally performed under anaerobic conditions using glycerol as the sole carbon source and in the absence of other exogenous reducing equivalent acceptors. Under these conditions, in e.g., strains of Citrobacter, Clostridium, and Klebsiella, a parallel pathway for glycerol operates which first involves oxidation of glycerol to dihydroxyacetone (DHA) by a NAD.sup.+-(or NAPD.sup.+-) linked glycerol dehydrogenase, Equation 3. The DHA, following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA kinase (Equation 4), Glycerol+NAD.sup.+.fwdarw.DHA+NADH+H.sup.+ (Equation 3) DHA+ATP.fwdarw.DHAP+ADP (Equation 4) becomes available for biosynthesis and for supporting ATP generation via e.g., glycolysis. In contrast to the 1,3-propanediol pathway, this pathway may provide carbon and energy to the cell and produces rather than consumes NADH.

In Klebsiella pneumoniae and Citrobacter freundii, the genes encoding the functionally linked activities of glycerol dehydratase (dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon. The dha regulon, in Klebsiella pneumoniae and Citrobacter freundii, also encompasses a gene encoding a transcriptional activator protein (dhaR). The dha regulons from Citrobacter and Klebsiella have been expressed in Escherichia coli and have been shown to convert glycerol to 1,3-propanediol.

Neither the chemical nor biological methods described above for the production of 1,3-propanediol are well suited for industrial scale production since the chemical processes are energy intensive and the biological processes are limited to relatively low titer from the expensive starting material, glycerol. These drawbacks could be overcome with a method requiring low energy input and an inexpensive starting material such as carbohydrates or sugars, or by increasing the metabolic efficiency of a glycerol process. Development of either method will require the ability to manipulate the genetic machinery responsible for the conversion of sugars to glycerol and glycerol to 1,3-propanediol.

Biological processes for the preparation of glycerol are known. The overwhelming majority of glycerol producers are yeasts but some bacteria, other fungi and algae are also known. Both bacteria and yeasts produce glycerol by converting glucose or other carbohydrates through the fructose-1,6-bisphosphate pathway in glycolysis or the Embden Meyerhof Parnas pathway, whereas, certain algae convert dissolved carbon dioxide or bicarbonate in the chloroplasts into the 3-carbon intermediates of the Calvin cycle. In a series of steps, the 3-carbon intermediate, phosphoglyceric acid, is converted to glyceraldehyde 3-phosphate which can be readily interconverted to its keto isomer dihydroxyacetone phosphate and ultimately to glycerol.

Specifically, the bacteria Bacillus licheniformis and Lactobacillus lycopersica synthesize glycerol, and glycerol production is found in the halotolerant algae Dunaliella sp. and Asteromonas gracilis for protection against high external salt concentrations. Similarly, various osmotolerant yeasts synthesize glycerol as a protective measure. Most strains of Saccharomyces produce some glycerol during alcoholic fermentation, and this can be increased physiologically by the application of osmotic stress. Earlier this century commercial glycerol production was achieved by the use of Saccharomyces cultures to which "steering reagents" were added such as sulfites or alkalis. Through the formation of an inactive complex, the steering agents block or inhibit the conversion of acetaldehyde to ethanol; thus, excess reducing equivalents (NADH) are available to or "steered" towards DHAP for reduction to produce glycerol. This method is limited by the partial inhibition of yeast growth that is due to the sulfites. This limitation can be partially overcome by the use of alkalis that create excess NADH equivalents by a different mechanism. In this practice, the alkalis initiated a Cannizarro disproportionation to yield ethanol and acetic acid from two equivalents of acetaldehyde.

The gene encoding glycerol-3-phosphate dehydrogenase (DAR1, GPD1) has been cloned and sequenced from S. diastaticus (Wang et al., J. Bact. 176, 7091 7095 (1994)). The DAR1 gene was cloned into a shuttle vector and used to transform E. coli where expression produced active enzyme. Wang et al. (supra) recognize that DAR1 is regulated by the cellular osmotic environment but do not suggest how the gene might be used to enhance 1,3-propanediol production in a recombinant microorganism.

Other glycerol-3-phosphate dehydrogenase enzymes have been isolated: for example, sn-glycerol-3-phosphate dehydrogenase has been cloned and sequenced from Saccharomyces cerevisiae (Larason et al., Mol. Microbiol. 10, 1101 (1993)) and Albertyn et al. (Mol. Cell. Biol. 14, 4135 (1994)) teach the cloning of GPD1 encoding a glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae. Like Wang et al. (supra), both Albertyn et al. and Larason et al. recognize the osmo-sensitivity of the regulation of this gene but do not suggest how the gene might be used in the production of 1,3-propanediol in a recombinant microorganism.

As with G3PDH, glycerol-3-phosphatase has been isolated from Saccharomyces cerevisiae and the protein identified as being encoded by the GPP1 and GPP2 genes (Norbeck et al., J. Biol. Chem. 271, 13875 (1996)). Like the genes encoding G3PDH, it appears that GPP2 is osmosensitive.

Although a single microorganism conversion of fermentable carbon source other than glycerol or dihydroxyacetone to 1,3-propanediol is desirable, it has been documented that there are significant difficulties to overcome in such an endeavor. For example, Gottschalk et al. (EP 373 230) teach that the growth of most strains useful for the production of 1,3-propanediol, including Citrobacter freundii, Clostridium autobutylicum, Clostridium butylicum, and Klebsiella pneumoniae, is disturbed by the presence of a hydrogen donor such as fructose or glucose. Strains of Lactobacillus brevis and Lactobacillus buchner, which produce 1,3-propanediol in co-fermentations of glycerol and fructose or glucose, do not grow when glycerol is provided as the sole carbon source, and, although it has been shown that resting cells can metabolize glucose or fructose, they do not produce 1,3-propanediol (Veiga DA Cunha et al., J. Bacteriol., 174, 1013 (1992)). Similarly, it has been shown that a strain of Ilyobacter polytropus, which produces 1,3-propanediol when glycerol and acetate are provided, will not produce 1,3-propanediol from carbon substrates other than glycerol, including fructose and glucose (Steib et al., Arch. Microbiol. 140, 139 (1984)). Finally, Tong et al. (Appl. Biochem. Biotech. 34, 149 (1992)) taught that recombinant Escherichia coli transformed with the dha regulon encoding glycerol dehydratase does not produce 1,3-propanediol from either glucose or xylose in the absence of exogenous glycerol.

Attempts to improve the yield of 1,3-propanediol from glycerol have been reported where co-substrates capable of providing reducing equivalents, typically fermentable sugars, are included in the process. Improvements in yield have been claimed for resting cells of Citrobacter freundii and Klebsiella pneumoniae DSM 4270 co-fermenting glycerol and glucose (Gottschalk et al., supra.; and Tran-Dinh et al., DE 3734 764); but not for growing cells of Klebsiella pneumoniae ATCC 25955 co-fermenting glycerol and glucose, which produced no 1,3-propanediol (I-T. Tong, Ph.D. Thesis, University of Wisconsin-Madison (1992)). Increased yields have been reported for the cofermentation of glycerol and glucose or fructose by a recombinant Escherichia coli; however, no 1,3-propanediol is produced in the absence of glycerol (Tong et al., supra.). In these systems, single microorganisms use the carbohydrate as a source of generating NADH while providing energy and carbon for cell maintenance or growth. These disclosures suggest that sugars do not enter the carbon stream that produces 1,3-propanediol.

Recently, however, the conversion of carbon substrates, other than glycerol or dihydroxyacetone, to 1,3-propanediol by a single microorganism that expresses a dehydratase enzyme has been described (U.S. Pat. No. 5,686,276; WO 9821339; WO 9928480; and WO 9821341 (U.S. Pat. No. 6,013,494)). A specific deficiency in the biological processes leading to the production of 1,3-propanediol from either glycerol or glucose has been the low titer of the product achieved via fermentation; thus, an energy-intensive separation process to obtain 1,3-propanediol from the aqueous fermentation broth is required. Fed batch or batch fermentations of glycerol to 1,3-propanediol have led to final titers of 65 g/L by Clostridium butyricum (Saint-Amans et al., Biotechnology Letters 16, 831 (1994)), 71 g/L by Clostridium butyricum mutants (Abbad-Andaloussi et al., Appl. Environ. Microbiol. 61, 4413 (1995)), 61 g/L by Klebsiella pneumoniae (Homann et al., Appl. Bicrobiol. Biotechnol. 33, 121 (1990)), and 35 g/L by Citrobacter freundii (Homann et al., supra). Fermentations of glucose to 1,3-propanediol that exceed the titer obtained from glycerol fermentations have not yet been disclosed.

The problem that remains to be solved is how to biologically produce 1,3-propanediol, with high titer and by a single microorganism, from an inexpensive carbon substrate such as glucose or other sugars. The biological production of 1,3-propanediol requires glycerol as a substrate for a two-step sequential reaction in which a dehydratase enzyme (typically a coenzyme B.sub.12-dependent dehydratase) converts glycerol to an intermediate, 3-hydroxypropionaldehyde, which is then reduced to 1,3-propanediol by a NADH- (or NADPH) dependent oxidoreductase. The complexity of the cofactor requirements necessitates the use of a whole cell catalyst for an industrial process that utilizes this reaction sequence for the production of 1,3-propanediol.

SUMMARY OF THE INVENTION

Applicants have solved the stated problem and the present invention provides for bioconverting a fermentable carbon source directly to 1,3-propanediol at significantly higher titer than previously obtained and with the use of a single microorganism. Glucose is used as a model substrate and E. coli is used as the model host. In one aspect of this invention, recombinant E. coli expressing a group of genes (comprising genes that encode a dehydratase activity, a dehydratase reactivation factor, a 1,3-propanediol oxidoreductase (dhaT), a glycerol-3-phosphate dehydrogenase, and a glycerol-3-phosphatase) convert glucose to 1,3-propanediol at titer that approaches that of glycerol to 1,3-propanediol fermentations.

In another aspect of this invention, the elimination of the functional dhaT gene in this recombinant E. coli results in a significantly higher titer of 1,3-propanediol from glucose. This unexpected increase in titer results in improved economics, and thus, an improved process for the production of 1,3-propanediol from glucose.

Furthermore, the present invention may be generally applied to include any carbon substrate that is readily converted to 1) glycerol, 2) dihydroxyacetone, 3) C.sub.3 compounds at the oxidation state of glycerol (e.g., glycerol 3-phosphate), or 4) C.sub.3 compounds at the oxidation state of dihydroxyacetone (e.g., dihydroxyacetone phosphate or glyceraldehyde 3-phosphate). The production of 1,3-propanediol in the dhaT minus strain requires a non-specific catalytic activity that converts 3-HPA to 1,3-propanediol. Identification of the enzyme(s) and/or gene(s) responsible for the non-specific catalytic activity that converts 3-HPA to 1,3-propanediol will lead to production of 1,3-propanediol in a wide range of host microorganisms with substrates from a wide range of carbon-containing substrates. It is also anticipated that the use of this non-specific catalytic activity that converts 3-HPA to 1,3-propanediol will lead to an improved process for the production of 1,3-propanediol from glycerol or dihydroxyacetone, by virtue of an improved titer and the resulting improved economics.

This activity has been isolated from E. coli as a nucleic acid fragment encoding a non-specific catalytic activity for the conversion of 3-hydroxypropionaldehyde to 1,3-propanediol, as set out in SEQ ID NO:58 or as selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence of SEQ ID NO:57; (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence of SEQ ID NO:57; (c) an isolated nucleic acid fragment encoding a polypeptide of at least 387 amino acids having at least 80% with the amino acid sequence of SEQ ID NO:57; (d) an isolated nucleic acid fragment that hybridizes with (a) under hybridization conditions of 0.1.times.SSC, 0.1% SDS, 65.degree. C. and washed with 2.times.SSC, 0.1% SDS followed by 0.1.times.SSC, 0.1% SDS; and (d) an isolated nucleic acid fragment that is complementary to (a), (b), (c), or (d). Alternatively, the nonspecific catalytic acitivity is embodied in the polypeptide as set out in SEQ ID NO:57.

A chimeric gene may be constructed comprising the isolated nucleic acid fragment described above operably linked to suitable regulatory sequences. This chimeric gene can be used to transform miciroorganisms selected from the group consisting of Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Salmonella, Bacillus, Aerobacter, Streptomyces, Escherichia, and Pseudomonas. E. coli is the preferred host.

Accordingly, the present invention provides a recombinant microorganism, useful for the production of 1,3-propanediol comprising: (a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; (b) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity; (c) at least one gene encoding a polypeptide having a dehydratase activity; (d) at least one gene encoding a dehydratase reactivation factor; (e) at least one endogenous gene encoding an non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol, wherein no functional dhaT gene encoding a 1,3-propanediol oxidoreductase is present. The preferred embodiment is a recombinant microorganism (preferably E. coli) where no dhaT gene is present. Optionally, the recombinant microorganism may comprise mutations (e.g., deletion mutations or point mutations) in endogenous genes selected from the group consisting of: (a) a gene encoding a polypeptide having glycerol kinase activity; (b) a gene encoding a polypeptide having glycerol dehydrogenase activity; and (c) gene encoding a polypeptide having triosephosphate isomerase activity.

In another embodiment the invention includes a process for the production of 1,3-propanediol comprising: (a) contacting, under suitable conditions, a recombinant E. coli comprising a dha regulon and lacking a functional dhaT gene encoding a 1,3-propanediol oxidoreductase activity with at least one carbon source, wherein the carbon source is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and single-carbon substrates; and (b) optionally recovering the 1,3-propanediol produced in (a).

The invention also provides a process for the production of 1,3-propanediol from a recombinant microorganism comprising: (a) contacting the recombinant microorganism of the present invention with at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and single-carbon substrates whereby 1,3-propanediol is produced; and (b) optionally recovering the 1,3-propanediol produced in (a).

Similarly the invention intends to provide a process for the production of 1,3-propanediol from a recombinant microorganism comprising:

(a) contacting a recombinant microorganism with at least one carbon source, said recombinant microorganism comprising: (i) at least one gene encoding a polypeptide having a dehydratase activity; (ii) at least one gene encoding a dehydratase reactivation factor; (iii) at least one endogenous gene encoding a non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol; wherein no functional dhaT gene encoding a 1,3-propanediol oxidoreductase is present; said carbon source selected from the group consisting of glycerol and dihydroxyacetone, wherein 1,3-propanediol is produced and;

(b) optionally recovering the 1,3-propanediol produced in (a).

Yet another aspect of the invention provides for the co-feeding of the carbon substrate. In this embodiment for the production of 1,3-propanediol, the steps are: (a) contacting a recombinant E. coli with a first source of carbon and with a second source of carbon, said recombinant E. coli comprising: (i) at least one exogenous gene encoding a polypeptide having a dehydratase activity; (ii) at least one exogenous gene encoding a dehydratase reactivation factor; (iii) at least one exogenous gene encoding a non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol, wherein no functional dhaT gene encoding a 1,3-propanediol oxidoreductase activity is present in the recombinant E. coli and wherein said first carbon source is selected from the group consisting of glycerol and dihydroxyacetone, and said second carbon source is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and single-carbon substrates, and (b) the 1,3-propanediol produced in (a) is optionally recovered. The co-feed may be sequential or simultaneous. The recombinant E. coli used in a co-feeding embodiments may further comprise: (a) a set of exogenous genes consisting of (i) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; (ii) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity; and (iii) at least one subset of genes encoding the gene products of dhaR, orfR, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ, and (b) a set of endogenous genes, each gene having a mutation inactivating the gene, the set consisting of: (i) a gene encoding a polypeptide having glycerol kinase activity; (ii) a gene encoding a polypeptide having glycerol dehydrogenase activity; and (iii) a gene encoding a polypeptide having triosephosphate isomerase activity.

Useful recombinant E. coli strains include recombinant E. coli strain KLP23 comprising: (a) a set of two endogenous genes, each gene having a mutation inactivating the gene, the set consisting of: (i) a gene encoding a polypeptide having a glycerol kinase activity; and (ii) a gene encoding a polypeptide having a glycerol dehydrogenase activity; (b) at least one exogenous gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; (c) at least one exogenous gene encoding a polypeptide having glycerol-3-phosphatase activity; and (d) a plasmid pKP32 and a recombinant E. coli strain RJ8 comprising: (a) set of three endogenous genes, each gene having a mutation inactivating the gene, the set consisting of: (i) a gene encoding a polypeptide having a glycerol kinase activity; (ii) a gene encoding a polypeptide having a glycerol dehydrogenase activity; and (iii) a gene encoding a polypeptide having a triosephosphate isomerase activity.

Other useful embodiments include recombinant E. coli comprising: (a) a set of exogenous genes consisting of: (i) at least one gene encoding a polypeptide having a dehydratase activity; (ii) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; (iii) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity; and (iv) at least one gene encoding a dehydratase reactivation factor; and (b) at least one endogenous gene encoding a non-specific catalytic activity to convert 3-hydroxypropionaldehyde to 1,3-propanediol; wherein no functional dhaT gene encoding a 1,3-propanediol oxidoreductase activity is present in the recombinant E. coli.

Another embodiments is a recombinant E. coli comprising: (a) a set of exogenous genes consisting of (i) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; (ii) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity; and (iii) at least one subset of genes encoding the gene products of dhaR, orfY, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ, and (b) at least one endogenous gene encoding a non-specific catalytic activity to convert 3-hydroxypropionaldehyde to 1,3-propanediol; wherein no functional dhaT gene encoding a 1,3-propanediol oxidoreductase activity is present in the recombinant E. coli. This embodiment also includes a process using a recombinant E. coli further comprising a set of endogenous genes, each gene having a mutation inactivating the gene, the set consisting of: (a) a gene encoding a polypeptide having glycerol kinase activity; (b) a gene encoding a polypeptide having glycerol dehydrogenase activity; and (c) a gene encoding a polypeptide having triosephosphate isomerase activity.

This embodiment still further includes a process for the bioproduction of 1,3-propanediol comprising: (a) contacting under suitable conditions the immediately disclosed recombinant E. coli with at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and single-carbon substrates whereby 1,3-propanediol is produced; and (b) optionally recovering the 1,3-propanediol produced in (a).

And also includes a further process for the bioproduction of 1,3-propanediol comprising: (a) contacting the recombinant E. coli of the immediately disclosed embodiments that further comprise: (i) at least one exogenous gene encoding a polypeptide having a dehydratase activity; (ii) at least one exogenous gene encoding a dehydratase reactivation factor; (iii) at least one endogenous gene encoding a non-specific catalytic activity to convert 3-hydroxypropionaldehyde to 1,3-propanediol, with at least one carbon source selected from the group consisting of glycerol and dihydroxyacetone, and (b) optionally recovering the 1,3-propanediol produced in (a).

BRIEF DESCRIPTION OF THE DRAWINGS, SEQUENCE DESCRIPTIONS, AND BIOLOGICAL DEPOSITS

The invention can be more fully understood from the following detailed description, Figures, the accompanying sequence descriptions, and biological deposits that form parts of this application.

FIG. 1 presents the gene organization within the sequence of the dha regulon subclone pHK28-26.

FIG. 2 presents a graph of the extracellular soluble protein (g/L) compared between two fermentations runs essentially as described in Example 7 using a constant feed of vitamin B.sub.12. In one case, solid lines, the strain used was KLP23/pAH48/pKP32. In the other case, dashed lines, the strain used was KLP23/pAH48/pDT29.

FIG. 3 presents a graph of the cell viability [(viable cells/mL)/OD550] compared between two fermentations runs essentially as described in Example 7 using a constant feed of vitamin B.sub.12. In one case (solid lines), the strain used was KLP23/pAH48/pKP32. In the other case (dashed lines), the strain used was KLP23/pAH48/pDT29.

FIG. 4 presents a graph of the yield of glycerol from glucose compared between two fermentations runs essentially as described in Example 7, but in the absence of vitamin B.sub.12 or coenzyme B.sub.12. In one case (solid lines), the strain used was KLP23/pAH48/pKP32. In the other case (dashed lines), the strain used was KLP23/pAH48/pDT29.

FIG. 5 is a flow diagram illustrating the metabolic conversion of glucose to 1,3-propanediol.

FIG. 6 is a 2D-PAGE membrane blot with the soluble protein fraction extracted from a band showing endogenous E. coli oxidoreductase activity (non-specific catalytic activity) on a native gel.

The 68 sequence descriptions and the sequence listing attached hereto will comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. .sctn.1.821 1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures--the Sequence Rules") and will be consistent with World Intellectual Property Organization (WIPO) Standard ST2.5 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administration Instructions). The Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IYUB standards described in Nucleic Acids Res. 13, 3021 3030 (1985) and in the Biochemical Journal 219, 345 373 (1984) which are herein incorporated by reference.

SEQ ID NO:1 contains the nucleotide sequence determined from a 12.1 kb EcoRI-SalI fragment from pKP1 (cosmid containing DNA from Klebsiella pneumoniae), subcloned into pIBI31 (IBI Biosystem, New Haven, Conn.), and termed pHK28-26. Table 1 further details genes, corresponding base pairs identified within SEQ ID NO:1, and associated functionality. See also Example 1.

SEQ ID NO:57 contains the nucleotide sequence determined for yqhD.

SEQ ID NO:58 contains the nucleotide sequence determined for yqhD.

Applicants have made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure:

TABLE-US-00001 Depositor Identification Int'l Depository Reference Designation Date of Deposit E. coli DH5.alpha.; transformed with ATCC 69789 18 Apr. 1995 plasmid pKP1 comprising a portion of the Klebsiella genome encoding the glycerol dehydratase enzyme E. coli DH5.alpha.; transformed with ATCC 69790 18 Apr. 1995 plasmid pKP4 comprising a portion of the Klebsiella genome encoding the diol dehydratase enzyme E. coli MSP33.6 comprising a ATCC 98598 25 Nov. 1997 deletion in gldA E. coli RJF10m comprising a ATCC 98597 25 Nov. 1997 deletion in glpK

The deposit(s) will be maintained in the indicated international depository for at least 30 years and will be made available to the public upon the grant of a patent disclosing it. The availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.

As used herein, "ATCC" refers to the American Type Culture Collection international depository located 10801 University Blvd., Manassas, Va. 20110-2209 U.S.A. The "ATCC No." is the accession number to cultures on deposit with the ATCC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for an improved process for bioconverting a fermentable carbon source directly to 1,3-propanediol using a single microorganism. The method is characterized by improved titer, yield, and cell viability as well as a decrease in cell lysis during fermentation.

The present invention is based, in part, upon the observation that 1,3-propanediol fermentation processes comprising 1,3-propanediol oxidoreductase (dhaT) are characterized by high levels of 3HPA and other aldehydes and ketones in the medium, which is correlated to a decrease in cell viability. The present invention is also based, in part, upon the unexpected finding that the model host, E. coli, is capable of converting 3-HPA to 1,3-propanediol by an endogenous non-specific catalytic activity capable of converting 3-hydroxypropionaldehyde to 1,3-propanediol. The present invention is further based, in part, upon the unexpected finding that an E. coli fermentation process comprising this non-specific catalytic activity and lacking a functional dhaT results in increased cell viability during fermentation and provides for higher titers and/or yields of 1,3-propanediol than a fermentation process comprising a functional dhaT.

In one aspect, glycerol is a model substrate, the host microorganism has a mutation in wild-type dhaT such that there is no 1,3-propanediol oxidoreductase activity and comprises a non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol. In another aspect, glucose is a model substrate and recombinant E. coli is a model host. In this aspect, E. coli comprises an endogenous non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol. In one embodiment, the non-specific catalytic activity is an alcohol dehydrogenase.

In one aspect, the present invention provides a recombinant E. coli expressing a group of genes comprising (a) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; (b) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity; (c) at least one gene encoding a polypeptide having a dehydratase activity; (d) at least one gene encoding a dehydratase reactivation factor; and (e) at least one endogenous gene encoding an non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol; use of this microorganism converts glucose to 1,3-propanediol at a high titer. In another aspect of this invention, the elimination of the functional dhaT gene in this recombinant E. coli provides an unexpectedly higher titer of 1,3-propanediol from glucose than previously attained.

The present invention provides an improved method for the biological production of 1,3-propanediol from a fermentable carbon source in a single microorganism. In one aspect of the present invention, an improved process for the conversion of glucose to 1,3-propanediol is achieved by the use of a recombinant microorganism comprising a host E. coli transformed with the Klebsiella pneumoniae dha regulon genes dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, and orfZ, all these genes arranged in the same genetic organization as found in wild type Klebsiella pneumoniae. The titer obtained for the fermentation process is significantly higher than any titer previously reported for a similar fermentation. This improvement relies on the use of the plasmid pDT29 as described in Example 6 and Example 7.

In another aspect of the present invention, a further improved process for the production of 1,3-propanediol from glucose is achieved using a recombinant E. coli containing genes encoding a G3PDH, a G3P phosphtase, a dehydratase, and a dehydratase reactivation factor compared to a process using a recombinant E. coli containing genes encoding a G3PDH, a G3P phosphatase, a dehydratase, a dehydratase reactivation factor, and also a functional dhaT. The dramatically improved process relies on an endogenous gene encoding a non-specific catalytic activity, expected to be an alcohol dehydrogenase, which is present in E. coli.

The dramatic improvement in the process is evident as an increase in 1,3-propanediol titer as illustrated in Examples 7 and 9. The improvement in the process is also evident as a decrease in cell lysis as determined by the extracellular soluble protein concentration in the fermentation broth. This aspect of the invention is illustrated in FIG. 2. Additionally, the improvement in the process is evident as prolonged cell viability over the course of the fermentation. This aspect of the invention is illustrated in FIG. 3. Furthermore, the improvement in the process is also evident as an increase in yield. In E. coli expressing a 1,3-propanediol oxidoreductase (dhaT) (for example, E. coli KLP23 transformed with the plasmid pDT29), glycerol can be metabolized to a product other than 3-HPA. In direct contrast, in E. coli not expressing a 1,3-propanediol oxidoreductase (dhaT) (for example, E. coli KLP23 transformed with the plasmid pKP32), glycerol is not metabolized to a product other than 3-HPA. That this cryptic pathway is attributable to the presence or absence of a functional dhaT is demonstrated by the lower yield of glycerol from glucose as illustrated in FIG. 4.

As used herein the following terms may be used for interpretation of the claims and specification.

The terms "glycerol-3-phosphate dehydrogenase" and "G3PDH" refer to a polypeptide responsible for an enzyme activity that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P). In vivo G3PDH may be NADH; NADPH; or FAD-dependent. When specifically referring to a cofactor specific glycerol-3-phosphate dehydrogenase, the terms "NADH-dependent glycerol-3-phosphate dehydrogenase", "NADPH-dependent glycerol-3-phosphate dehydrogenase" and "FAD-dependent glycerol-3-phosphate dehydrogenase" will be used. As it is generally the case that NADH-dependent and NADPH-dependent glycerol-3-phosphate dehydrogenases are able to use NADH and NADPH interchangeably (for example by the gene encoded by gpsA), the terms NADH-dependent and NADPH-dependent glycerol-3-phosphate dehydrogenase will be used interchangeably. The NADH-dependent enzyme (EC 1.1.1.8) is encoded, for example, by several genes including GPD1 (GenBank Z74071x2), or GPD2 (GenBank Z35169x1), or GPD3 (GenBank G984182), or DAR1 (GenBank Z74071x2). The NADPH-dependent enzyme (EC 1.1.1.94) is encoded by gpsA (GenBank U321643, (cds 197911-196892) G466746 and L45246). The FAD-dependent enzyme (EC 1.1.99.5) is encoded by GUT2 (GenBank Z47047x23), or glpD (GenBank G147838), or glpABC (GenBank M20938) (see WO 9928480 and references therein, which are herein incorporated by reference).

The terms "glycerol-3-phosphatase", "sn-glycerol-3-phosphatase", or "d,l-glycerol phosphatase", and "G3P phosphatase" refer to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol-3-phosphate and water to glycerol and inorganic phosphate. G3P phosphatase is encoded, for example, by GPP1 (GenBank Z47047x125), or GPP2 (GenBank U18813x11) (see WO 9928480 and references therein, which are herein incorporated by reference).

The term "glycerol kinase" refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol and ATP to glycerol-3-phosphate and ADP. The high-energy phosphate donor ATP may be replaced by physiological substitutes (e.g., phosphoenolpyruvate). Glycerol kinase is encoded, for example, by GUT1 (GenBank U11583x19) and glpK (GenBank L19201) (see WO 9928480 and references therein, which are herein incorporated by reference).

The term "glycerol dehydrogenase" refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol to dihydroxyacetone (E.C. 1.1.1.6) or glycerol to glyceraldehyde (E.C. 1.1.1.72). A polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol to dihydroxyacetone is also referred to as a "dihydroxyacetone reductase". Glycerol dehydrogenase may be dependent upon NADH (E.C. 1.1.1.6), NADPH (E.C. 1.1.1.72), or other cofactors (e.g., E.C. 1.1.99.22). A NADH-dependent glycerol dehydrogenase is encoded, for example, by gldA (GenBank U00006) (see WO 9928480 and references therein, which are herein incorporated by reference).

The term "dehydratase enzyme" or "dehydratase" will refer to any enzyme activity that catalyzes the conversion of a glycerol molecule to the product 3-hydroxypropionaldehyde. For the purposes of the present invention the dehydratase enzymes include a glycerol dehydratase (E.C. 4.2.1.30) and a diol dehydratase (E.C. 4.2.1.28) having preferred substrates of glycerol and 1,2-propanediol, respectively. Genes for dehydratase enzymes have been identified in Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Salmonella typhimurium, and Klebsiella oxytoca. In each case, the dehydratase is composed of three subunits: the large or ".alpha." subunit, the medium or ".beta." subunit, and the small or ".gamma." subunit. Due to the wide variation in gene nomenclature used in the literature, a comparative chart is given in Table 1 to facilitate identification. The genes are also described in, for example, Daniel et al. (FEMS Microbiol. Rev. 22, 553 (1999)) and Toraya and Mori (J. Biol. Chem. 274, 3372 (1999)). Referring to Table 1, genes encoding the large or ".alpha." subunit of glycerol dehydratase include dhaB1, gldA and dhaB; genes encoding the medium or ".beta." subunit include dhaB2, gldB, and dhaC; genes encoding the small or ".gamma." subunit include dhaB3, gldC, and dhaE. Also referring to Table 1, genes encoding the large or ".alpha." subunit of diol dehydratase include pduC and pddA; genes encoding the medium or ".beta." subunit include pduD and pddB; genes encoding the small or ".gamma." subunit include pduE and pddC.

TABLE-US-00002 TABLE 1 Comparative chart of gene names and GenBank references for dehydratase and dehydratase linked functions. GENE FUNCTION: 1,3-PD ORGANISM regulatory unknown reactivation dehydrogenase unknown (GenBank Reference) gene base pairs gene base pairs geme base pairs gene base pairs gene base pairs K. pneumoniae dhaR 2209 4134 orfW 4112 4642 orfX 4643 4996 dhaT 5017 6108 orfY 6202 6630 (SEQ ID NO: 1) K. pneumoniae (U30903) orf2c 7116 7646 orf2b 6762 7115 dhaT 5578 6741 orf2a 5125 5556 K. pneumoniae (U60992) gdrB C. freundii (U09771) dhaR 3746 5671 orfW 5649 6179 orfX 6180 6533 dhaT 6550 7713 orfY 7736 8164 C. pasteurianum (AF051373) C. pasteurianum (AF006034) orfW 210 731 orfX 1 196 dhaT 1232 2389 orfY 746 1177 S. typhimurium (AF026270) pduH 8274 8645 K. oxytoca (AF017781) ddrB 2063 2440 K. oxytoca (AF051373) GENE FUNCTION: ORGANISM dehydratase, .alpha. dehydratase, .beta. dehydratase, .gamma. reactivation (GenBank Reference) gene base pairs gene base pairs gene base pairs gene base pairs K. pneumoniae dhaB1 7044 8711 dhaB2 8724 9308 dhaB3 9311 9736 orfZ 9749 11572 (SEQ ID NO: 1) K. pneumoniae (U30903) dhaB1 3047 4714 dhaB2 2450 2890 dhaB3 2022 2447 dhaB4 186 2009 K. pneumoniae (U60992) gldA 121 1788 gldB 1801 2385 gldC 2388 2813 gdrA C. freundii (U09771) dhaB 8556 10223 dhaC 10235 10819 dhaE 10822 11250 orfZ 11261 13072 C. pasteurianum (AF051373) dhaB 84 1748 dhaC 1779 2318 dhaE 2333 2773 orfZ 2790 4598 C. pasteurianum (AF006034) S. typhimurium (AF026270) pduC 3557 5221 pduD 5232 5906 pduE 5921 6442 pduG 6452 8284 K. oxytoca (AF017781) ddrA 241 2073 K. oxytoca (AF051373) pddA 121 1785 pddB 1796 2470 pddC 2485 3006

Glycerol and diol dehydratases are subject to mechanism-based suicide inactivation by glycerol and some other substrates (Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999)). The term "dehydratase reactivation factor" refers to those proteins responsible for reactivating the dehydratase activity. The terms "dehydratase reactivating activity", "reactivating the dehydratase activity" or "regenerating the dehydratase activity" refers to the phenomenon of converting a dehydratase not capable of catalysis of a substrate to one capable of catalysis of a substrate or to the phenomenon of inhibiting the inactivation of a dehydratase or the phenomenon of extending the useful half-life of the dehydratase enzyme in vivo. Two proteins have been identified as being involved as the dehydratase reactivation factor (see WO 9821341 (U.S. Pat. No. 6,013,494) and references therein, which are herein incorporated by reference; Daniel et al., supra; Toraya and Mori, J. Biol. Chem. 274, 3372 (1999); and Tobimatsu et al., J. Bacteriol. 181, 4110 (1999)). Referring to Table 1, genes encoding one of the proteins include orfZ, dhaB4, gdrA, pduG and ddrA. Also referring to Table 1, genes encoding the second of the two proteins include orfX, orf2b, gdrB, pduH and ddrB.

The terms "1,3-propanediol oxidoreductase", "1,3-propanediol dehydrogenase" or "DhaT" refer to the polypeptide(s) responsible for an enzyme activity that is capable of catalyzing the interconversion of 3-HPA and 1,3-propanediol provided the gene(s) encoding such activity is found to be physically or transcriptionally linked to a dehydratase enzyme in its natural (i.e., wild type) setting; for example, the gene is found within a dha regulon as is the case with dhaT from Klebsiella pneumonia. Referring to Table 1, genes encoding a 1,3-propanediol oxidoreductase include dhaT from Klebsiella pneumoniae, Citrobacter freundii, and Clostridium pasteurianum. Each of these genes encode a polypeptide belonging to the family of type III alcohol dehydrogenases, exhibits a conserved iron-binding motif, and has a preference for the NAD.sup.+/NADH linked intercoversion of 3-HPA and 1,3-propanediol (Johnson and Lin, J. Bacteriol. 169, 2050 (1987); Daniel et al., J. Bacteriol. 177, 2151 (1995); and Leurs et al., FEMS Microbiol. Lett. 154, 337 (1997)). Enzymes with similar physical properties have been isolated from Lactobacillus brevis and Lactobacillus buchneri (Veiga da Dunha and Foster, Appl. Environ. Microbiol. 58, 2005 (1992)).

The term "dha regulon" refers to a set of associated genes or open reading frames encoding various biological activities, including but not limited to a dehydratase activity, a reactivation activity, and a 1,3-propanediol oxidoreductase. Typically a dha regulon comprises the open reading frames dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, and orfZ as described herein.

The term "non-specific catalytic activity" refers to the polypeptide(s) responsible for an enzyme activity that is sufficient to catalyze the interconversion of 3-HPA and 1,3-propanediol and specifically excludes 1,3-propanediol oxidoreductase(s). Typically these enzymes are alcohol dehydrogenases. Such enzymes may utilize cofactors other than NAD.sup.+/NADH, including but not limited to flavins such as FAD or FMN. A gene(s) for a non-specific alcohol dehydrogenase(s) is found, for example, to be endogenously encoded and functionally expressed within the microorganism E. coli KLP23.

The terms "function" or "enzyme function" refer to the catalytic activity of an enzyme in altering the energy required to perform a specific chemical reaction. It is understood that such an activity may apply to a reaction in equilibrium where the production of either product or substrate may be accomplished under suitable conditions.

The terms "polypeptide" and "protein" are used interchangeably.

The terms "carbon substrate" and "carbon source" refer to a carbon source capable of being metabolized by host microorganisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.

The terms "host cell" or "host microorganism" refer to a microorganism capable of receiving foreign or heterologous genes and of expressing those genes to produce an active gene product.

The terms "foreign gene", "foreign DNA", "heterologous gene" and "heterologous DNA" refer to genetic material native to one organism that has been placed within a host microorganism by various means. The gene of interest may be a naturally occurring gene, a mutated gene, or a synthetic gene.

The terms "transformation" and "transfection" refer to the acquisition of new genes in a cell after the incorporation of nucleic acid. The acquired genes may be integrated into chromosomal DNA or introduced as extrachromosomal replicating sequences. The term "transformant" refers to the product of a transformation.

The term "genetically altered" refers to the process of changing hereditary material by transformation or mutation.

The terms "recombinant microorganism" and "transformed host" refer to any microorganism having been transformed with heterologous or foreign genes or extra copies of homologous genes. The recombinant microorganisms of the present invention express foreign genes encoding glycerol-3-phosphate dehydrogenase (GPD1), glycerol-3-phosphatase (GPP2), glycerol dehydratase (dhaB1, dhaB2 and dhaB3), dehydratase reactivation factor (orfZ and orfX), and optionally 1,3-propanediol oxidoreductase (dhaT) for the production of 1,3-propanediol from suitable carbon substrates. A preferred embodiment is an E. coli transformed with these genes but lacking a functional dhaT. A host microorganism, other than E. coli, may also be transformed to contain the disclosed genes and the gene for the non-specific catalytic activity for the interconversion of 3-HPA and 1,3-propanediol, specifically excluding 1,3-propanediol oxidoreductase(s) (dhaT).

"Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding) and following (3' non-coding) the coding region. The terms "native" and "wild-type" refer to a gene as found in nature with its own regulatory sequences.

The terms "encoding" and "coding" refer to the process by which a gene, through the mechanisms of transcription and translation, produces an amino acid sequence. It is understood that the process of encoding a specific amino acid sequence includes DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary sequences.

The term "isolated" refers to a protein or DNA sequence that is removed from at least one component with which it is naturally associated.

An "isolated nucleic acid molecule" is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

"Substantially similar" refers to nucleic acid molecules wherein changes in one or more nucleotide bases result in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. "Substantially similar" also refers to nucleic acid molecules wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid molecule to mediate alteration of gene expression by antisense or co-suppression technology. "Substantially similar" also refers to modifications of the nucleic acid molecules of the instant invention (such as deletion or insertion of one or more nucleotide bases) that do not substantially affect the functional properties of the resulting transcript vis-a-vis the ability to mediate alteration of gene expression by antisense or co-suppression technology or alteration of the functional properties of the resulting protein molecule. The invention encompasses more than the specific exemplary sequences.

For example, it is well known in the art that alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded protein are common. For the purposes of the present invention substitutions are defined as exchanges within one of the following five groups: 1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly); 2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln; 3. Polar, positively charged residues: His, Arg, Lys; 4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and 5. Large aromatic residues: Phe, Tyr, Trp.

Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes which result in substitution of one negatively charged residue for another (such as aspartic acid for glutamic acid) or one positively charged residue for another (such as lysine for arginine) can also be expected to produce a functionally equivalent product.

In many cases, nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.

Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Moreover, the skilled artisan recognizes that substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1.times.SSC, 0.1% SDS, 65.degree. C. and washed with 2.times.SSC, 0.1% SDS followed by 0.1.times.SSC, 0.1% SDS), with the sequences exemplified herein. Preferred substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are at least 80% identical to the DNA sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are at least 90% identical to the DNA sequence of the nucleic acid fragments reported herein. Most preferred are nucleic acid fragments that are at least 95% identical to the DNA sequence of the nucleic acid fragments reported herein.

A nucleic acid fragment is "hybridizable" to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference).